CRISPR screening identifies the deubiquitylase ATXN3 as a PD-L1–positive regulator for tumor immune evasion

Regulation of tumoral PD-L1 expression is critical to advancing our understanding of tumor immune evasion and the improvement of existing antitumor immunotherapies. Herein, we describe a CRISPR-based screening platform and identified ATXN3 as a positive regulator for PD-L1 transcription. TCGA database analysis revealed a positive correlation between ATXN3 and CD274 in more than 80% of human cancers. ATXN3-induced Pd-l1 transcription was promoted by tumor microenvironmental factors, including the inflammatory cytokine IFN-γ and hypoxia, through protection of their downstream transcription factors IRF1, STAT3, and HIF-2α. Moreover, ATXN3 functioned as a deubiquitinase of the AP-1 transcription factor JunB, indicating that ATNX3 promotes PD-L1 expression through multiple pathways. Targeted deletion of ATXN3 in cancer cells largely abolished IFN-γ– and hypoxia-induced PD-L1 expression and consequently enhanced antitumor immunity in mice, and these effects were partially reversed by PD-L1 reconstitution. Furthermore, tumoral ATXN3 suppression improved the preclinical efficacy of checkpoint blockade antitumor immunotherapy. Importantly, ATXN3 expression was increased in human lung adenocarcinoma and melanoma, and its levels were positively correlated with PD-L1 as well as its transcription factors IRF1 and HIF-2α. Collectively, our study identifies what we believe to be a previously unknown deubiquitinase, ATXN3, as a positive regulator for PD-L1 transcription and provides a rationale for targeting ATXN3 to sensitize checkpoint blockade antitumor immunotherapy.


Figure S1 .
Figure S1.CRISPR screening for PD-L1 deub regulators.(A) Schematic showing the lentiCRISPRv2 vector system used to generate the CRISPR-KO library.(B-D) Gating strategy for live cells (B).Representative Flow Cytometry plots confirming lentivirus infection of B16 cells through intracellular staining of Cas9 (C).Representative FACS plots from sorting.B16 cells were sorted on live cells followed by their PD-L1 expression.Top and bottom 5% of PD-L1 MFI populations were gated and sorted (D).(E-H) The RRA scores, p values and folds of enrichment of the identified guides are shown.

Figure s2 .
Figure s2.Analysis of PD-L1 expression on WT and ATXN3 KO cells.The expression levels of PD-L1 on WT and ATXN3 KO B16 melanoma (A) 4T1 triple-negative breast cancer (B) and MC38 colon cancer (C) cells was analyzed by flow cytometry.Representative images (left) and data from 3 repeated experiments (right) are shown.Two-tailed unpaired t test was performed to determine statistical significance.*** P < 0.001.

Figure
Figure s4.(A) Schematic showing modified Luciferase expression vector with luciferase expression under control of CD274 promoter used in (B-C).(B) Effect of ATXN3 on PD-L1 transcription was analyzed through dual-luciferase reporter assays.(C) Luciferase activity of PD-L1 reporter in HEK293T cells after co-transfection with different doses of ATXN3 plasmid.Ordinary one-way ANOVA with multiple comparisons was performed to determine significance.*P<0.05,**P<0.01,***P<0.001.

Figure s5 .
Figure s5.The effect of IRF1/STAT3 or HIF2a expression on PD-L1 expression in ATXN3 WT and KO LLC1 cells.WT or ATXN3 KO LLC1 cells were transfected with IRF1 and STAT3 (A) or with HIF-2a (B).36 hours later cells were cultivated either with IFN-g or under hypoxia condition for additional 24 hours and their surface PD-L1 expression were analyzed by flow cytometry.Ordinary one-way ANOVA with multiple comparisons was performed to determine significance.*P < 0.05, ** P < 0.01,*** P < 0.001.

Figure s6 .
Figure s6.ATXN3 is a JunB deubiquitinase.(A) ATXN3 interacts with JunB.Myc-ATXN3 expression plasmid was co-transfected with or without Flag-JunB into HEK293T cells.JunB protein was immunoprecipitated with anti-Flag antibody, bound ATXN3 was detected with anti-Myc antibody (top panel).(B) The endogenous interaction between ATXN3 and JunB in A549 cells.(C) HA-Ub and Flag-JunB expression plasmids were cotransfected with Myc-ATXN3 into HEK293T cells.JunB ubiquitination was determined by immunoprecipitation of JunB with anti-Flag antibodies and immunoblotting with HA antibody.(D & E) Flag-JunB was cotransfected with or without Myc-ATXN3 plasmids into HEK293T cells.The transfected cells were treated with cycloheximide (CHX) for different times.The protein levels of JunB (top panel) and ATXN3 (middle panel) were analyzed by western blotting.β-actin was used as a loading control (bottom).(E) Two-tailed unpaired t test was performed to determine statistical significance.*P<0.05,**P<0.01,***P<0.001.

Figure s8 .
Figure s8.ATXN3 inhibition improves anti-B16 melanoma immunity partially through downregulating tumoral PD-L1 expression.(A) WT or ATXN3 KO B16 melanoma cells were injected subcutaneously into C57BL/6 mice and then treated with or without anti-CD8 depletion Ab as indicated.Tumor growth curve was measured every 2 days.(C) Quantification of CD45 - cell-surface PD-L1 MFI from LLC1 tumors (n = 5).(D-H), Quantification of CD4 + T cells (D), CD8 + T cells (E) and Treg cells (F) percentage in CD45 + populations from B16 tumors as well as the CD8 T cell production of IFN-g (G) and Granzyme B (H) were analyzed.(A), (I) WT or ATXN3 KO B16 melanoma cells were injected and then treated with anti-PD-1 as indicated.(A

Figure s9 .
Figure s9.The effects of ATXN3 knockout on cancer cell growth.(A-C) The effect of Atxn3 knockout on LLC1 proliferation (A) and colony formation (B) in vitro were determined by WST-1 reagent and argar culture, respectively.The tumor progression of WT and ATXN3-KO LLC1 tumors in immune comprimized mice (C).(D-G) The effect of Atxn3 knockout on B16 (C & D) and